Many physiological events including embryogenesis, organ development, estrus, and wound healing require vascular growth and remodeling (Folkman et al., (1992) J. Biol. Chem. 267, 10931-10934; Risau, W. (1995) FASEB J. 9, 926-933.). In addition to these beneficial processes, angiogenesis is also involved in the proliferation of disease states such as tumor growth, metastasis, psoriasis, rheumatoid arthritis, macular degeneration and retinopathy (Pepper, M. S., (1996) Vasc. Med. 1, 259-266; Kuiper et al., (1998) Pharmacol. Res. 37, 1-16, 1998; Kumar and Fidler, (1998) In Vivo18, 27-34; Szekanecz et al., (1998) J. Investig. Med. 46, 27-41; Tolentino and Adamis, (1988) Int. Ophthalmol. Clin. 38, 77-94. Of the signaling pathways known to influence vascular formation, these involving vascular endothelial growth factor (VEGF) have been shown to be essential and selective for vascular endothelial cells (Dvorak et al., (1995) Am. J. Path. 146, 1029-1039; Thomas, K., (1996) J. Biol. Chem. 271, 603-606; Ferrara N. and Davis-Smyth, (1997) Endocrine Rev. 18, 4-25). The therapeutic potential of inhibiting the VEGF pathway has been directly demonstrated by anti-VEGF monoclonal antibodies which were active against a variety of human tumors (Borgstrom et al, (1996) Cancer Res. 56, 4032-4039) and ischemic retinal disease (Adamis et al., (1996) Arch. Ophthalmol. 114, 66-71).
Normal vasculogenesis and angiogenesis play important roles in a variety of physiological processes such as embryonic development, wound healing, organ regeneration and female reproductive processes such as follicle development in the corpus luteum during ovulation and placental growth after pregnancy (Folkman & Shing, 1992). Uncontrolled vasculogenesis and/or angiogenesis has been associated with diseases, such as diabetes, as well as malignant solid tumors that rely on vascularization for growth. Klagsburn & Soker, (1993) Current Biology 3(10):699-702; Folkham, (1991) J. Natl., Cancer Inst. 82:4-6; Weidner, et al., (1991) New Engl. J. Med. 324:1-5.
Several polypeptides with in vitro endothelial cell growth promoting activity have been identified. Examples include acidic and basic fibroblastic growth factor (FGF), vascular endothelial growth factor (VEGF)and placental growth factor. Unlike FGF, VEGF has recently been reported to be an endothelial cell specific mitogen (Ferrara & Henzel, (1989) Biochem. Biophys. Res. Comm. 161:851-858; Vaisman et al., (1990) J. Biol. Chem. 265:19461-19566).
Thus, identification of the specific receptors to which VEGF binds is important to understanding of the regulation of endothelial cell proliferation. Two structurally related tyrosine kinases have been identified to bind VEGF with high affinity: the fit-1 receptor (Shibuya et al., (1990) Oncogene 5:519-524; De Vries et al., (1992) Science 255:989-991) and the KDR/FLK-1receptor, discussed herein. Consequently, it had been surmised that RTKs may have a role in the modulation and regulation of endothelial cell proliferation.
Recent disclosures, such as information set forth in U.S. patent application Ser. Nos. 08/193,829, 08/038,596 and 07/975,750, strongly suggest that VEGF is not only responsible for endothelial cell proliferation, but also is the prime regulator of normal and pathological angiogenesis. See generally, Klagsbum & Soker, (1993) Current Biology 3:699-702; Houck, et al., (1992) J. Biol. Chem 267:26031-26037.
VEGF is a homodimeric cytokine that is expressed in at least four splice-variant forms of 121-206 residues (Ferrara and Davis-Smyth, 1997). Vascular endothelial cells express at least two high-affinity receptors for VEGF: VEGF-R1/Flt-1 and VEGFR-2/KDR. VEGF-R1 and VEGFR-2 are receptor tyrosine kinases each comprised of an extracellular domain that contains 7 immunoglobulin-like segments and binds VEGF, a short membrane spanning region, and a cytosolic domain possessing tyrosine kinase activity. The kinase domain directly follows the extracellular and juxtamembrane regions and is itself followed by another domain (post-kinase domain), which may function in binding of other proteins for signal transduction. These two receptors appear to have different signaling pathways and functions with VEGFR-2 being of primary importance in mitosis of endothelial cells (Waltenberger et al., (1994) J. Biol. Chem. 269, 26988-26995; Seetharm et al., (1995) Oncogene 10, 135-147; Shalaby et al., (1995) Nature 376, 576-579).
Both FGF and VEGF are potent angiogenic factors which induce formation of new capillary blood vessels. Transfection of human breast carcinoma cell line MCF-7 with FGF resulted in cell lines that form progressively growing and metastatic tumors when injected (s.c.) into nude mice. FGF may play a critical role in progression of breast tumors to an estrogen-independent, anti-estrogen resistant metastatic phenotype (McLeskey et al., (1993) Cancer Res. 53: 2168-2177). Breast tumor cells exhibited increased neovascularization, increased spontaneous metastasis and more rapid growth in vivo than did the non-transfected tumors. FGF has been shown to be transforming in NIH-3T3 cells and implicated in tumorigenesis and metastasis of mouse mammary tumors. FGF overexpression conferred a tumorigenic phenotype on a human adrenal carcinoma cell line suggesting that FGF's may also play a role in the transformation of epithelial cells. Polyclonal neutralizing antibodies to FGF inhibited tumor growth in Balb/c nude mice transplanted with K1000 cells (transfected with the leader sequence of bFGF) which form tumors in these mice (Hori et al., (1991) Cancer Res. 51: 6180-9184).
Due to the role of FGF in neovascularization, tumorigenesis and metastasis, there is a need in the art for FGF inhibitors as potent anti-cancer agents that exert their anti-FGF activity by preventing intracellular signaling of FGF.
VEGF, by contrast, is an endothelial cell-specific mitogen and an angiogenesis inducer that is released by a variety of tumor cells and expressed in human tumor cells in situ. Unlike FGF, transfection of cell lines with a cDNA sequence encoding VEGF, did not promote transformation, but did facilitate tumor growth in vivo (Ferrara, N., and Davis-Smyth, T. (1997)). Furthermore, administration of a polyclonal antibody which neutralized VEGF also inhibited growth of human rhabdomyosarcoma, glioblastoma multiforme and leiomyosarcoma cell lines in nude mice (Kim et al., (1993) Nature 362: 841-843).
In view of the importance of receptor tyrosine kinases (RTKs) to the control, regulation and modulation of endothelial cell proliferation and potentially vasculogenesis and/or angiogenesis, many attempts have been made to identify RTK "inhibitors" using a variety of approaches, including the use of mutant ligands (U.S. Pat. No. 4,966,849), soluble receptors and antibodies (Application No. WO 94/10202; Kendall & Thomas, (1994) Proc. Natl. Acad. Sci. 90:10705-09; Kim, et al., 1993), RNA ligands (Jellinek, et al., (1994) Biochemistry 3:10450-56), protein kinase C inhibitors (Schuchter, et al., (1991) Cancer Res. 51:682-687); Takano, et al., (1993) Mol. Bio. Cell 4:358A; Kinsella, et al., (1992) Exp. Cell Res. 199:56-62; Wright, et al., (1992) J. Cellular Phys. 152:448-57) and tyrosine kinase inhibitors (WO 94/03427; WO 92/21660; WO 91/15495; WO 94/14808; U.S. Pat. No. 5,330,992; Mariani, et al., (1994) Proc. Am. Assoc. Cancer Res. 35:2268).
More recently, attempts have been made to identify small molecules which act as tyrosine kinase inhibitors. For example, bis monocyclic, bicyclic or heterocyclic aryl compounds (PCT WO 92/20642), vinylene-azaindole derivatives (PCT WO 94/14808) and 1-cycloproppyl-4-pyridyl-quinolones (U.S. Pat. No. 5,330,992) have been described generally as tyrosine kinase inhibitors. Styryl compounds (U.S. Pat. No. 5,217,999), styryl-substituted pyridyl compounds (U.S. Pat. No. 5,302,606), certain quinazoline derivatives (EP Application No. 0 566 266 Al), selenoindoles and selenides (PCT WO 94/03427), tricyclic polyhydroxylic compounds (PCT WO 92/21660) and benzylphosphonic acid compounds (PCT WO 91/15495) have been described as compounds for use as tyrosine kinase inhibitors for use in the treatment of cancer. None of these compounds, however, have been previously associated with the enzymatic function of the VEGFR-2 receptor. Likewise, none of these compounds have been associated with regulation of vasculogenesis and/or angiogenesis.
Therefore, there is a need in the art to develop small molecule antagonists of the PDGF, FGF, EGF and VEGF pathways individually or as a group. Moreover, if these cytokines signal through a common second messenger pathway within the cell, such antagonists will have broad therapeutic activity to treat or prevent the progression of a broad array of diseases, such as coronary restenosis, tumor-associated angiogenesis, atherosclerosis, autoimmune diseases, acute inflammation, certain kidney diseases associated with proliferation of glomerular or mesangial cells, and ocular diseases associated with retinal vessel proliferation. The present invention was made by discovering a common signaling mechanism, a group of active therapeutic agents, shown to be active by a large number of and variety of predictive assays, and discovering a common intracellular signaling intermediate.
Based on sequence homology and overall domain structure, VEGFRs belong to the platelet-derived growth factor receptor family (PDGFR) which also includes PDGFR.alpha., PDGFR.beta., the stem cell growth factor receptor (c-kit), and the colony stimulating factor-1 receptor (CSF-1 R/c-fms) (van der Geer et al., (1994) Ann. Rev. Cell Biol. 10, 251-337). Compared to other protein kinases, members of this family contain an insert of approximately 65-97 residues, termed the kinase insert domain (KID), within the catalytic kinase domain relative to other protein kinases. Within the PDGFR family the KIDs are of varying length and low sequence homology. Deletion or mutation of the KID from PDGFR.alpha., PDGFR.beta., c-kit, and CSF-1 R have indicated that this domain is not necessary for intrinsic kinase activity but that it is important for the binding of other proteins involved in signal transduction, via autophosphorylation of KID tyrosine residues (Taylor et al., (1989) EMBO J. 8, 2029-2037; Heidaran et al., (1991) Mol. Cell. Biol. 11, 134-142; Yu et al., (1991) Mol. Cell. Biol. 11, 3780-3785; Kazlauskas et al., (1992) Mol. Cell. Biol. 12, 2534-2544; Lev et al., (1992) Proc. Natl. Acad. Sci. USA 89, 678-682; Reedjik et al., (1992) EMBO J. 11, 1365-1372; Bazenet et al., (1996) Mol. Cell. Biol. 16, 6926-6936). Although the signaling pathways and the specific role of the KID are still not fully determined for VEGFRs, the VEGFR-2 KID does contain two tyrosines which are known to be autophosphorylation sites (Dougher-Vermazen et al., (1994) Biochem. Biophys. Res. Comm. 205, 728-738).
Since the determination of the first cyclic AMP-dependent protein kinase (cAPK) structure (Knighton et al., (1991) Science 253, 407-413) a variety of protein kinase structures have been reported (reviewed in Johnson et al., (1996) Cell 85,149-158). Among the receptor protein lyrosine kinases (RTKs), structures of the kinase domain of the insulin receptor (IRK) (Hubbard, et al., (1994) Nature 372, 746-754; Hubbard, (1997) EMBO J. 16, 5572-5581) and the fibroblast growth factor receptor-1 (FGFR1) (Mohammadi et al., (1996) Cell 86, 577-87; Mohammadi et al., (1997) Science 276, 955-960) have been determined.